Balaenoptera physalus), that produce different call types associated with different behaviours, several hypotheses have emerged regarding call function, but the topic still remains in its infancy. Here, we investigate the potential function of two fin whale vocalizations, the song-forming 20-Hz call and the 40-Hz call, by examining their production in relation to season, year and prey biomass. Our results showed that the production of 20-Hz calls was strongly influenced by season, with a clear peak during the breeding months, and secondarily by year, likely due to changes in whale abundance. These results support the reproductive function of the 20-Hz song used as an acoustic display. Conversely, season and year had no effect on variation in 40-Hz calling rates, but prey biomass did. This is the first study linking 40-Hz call activity to prey biomass, supporting the previously suggested food-associated function of this call. Understanding the functions of animal signals can help identifying functional habitats and predict the negative effects of human activities with important implications for conservation.Animals use varied acoustic signals that play critical roles in their lives. Understanding the function of these signals may inform about key life-history processes relevant for conservation. In the case of fin whales ( These signals can encode various types of information about the signaller's attributes or external environment, and serve various purposes. During the mating season, males of many species produce high intensity and repetitive songs to attract or court females, repel conspecific males, or both –3. It haBalaenoptera physalus) produce distinct vocalizations but knowledge about the functions of their calls is still limited. The most reported fin whale call worldwide is the 20-Hz note [Fin whales -Hz note . Songs h-Hz note  and are -Hz note ,24. Thus-Hz note  or by ad-Hz note . Non-son-Hz note ,25. Fin -Hz note ,27. The -Hz note , in asso-Hz note ,29 and fTursiops truncatus) bray calls, humpback whale (Megaptera novaeangliae) ‘megapclicks’) [To investigate the hypothesized fin whale call functions, we examine variation in production rates of song-forming 20-Hz calls and 40-Hz calls with respect to season, year and prey biomass. If males use 20-Hz calls to attract females through acoustic display , we expeclicks’) ,31. To tclicks’) ,33. This. 2 (a)a) using ecological acoustic recorders (EARs) [Passive acoustic monitoring (PAM) data were collected at two locations off the Azores Archipelago a using es (EARs)  deployedb) and the 40-Hz call, a 0.3 s downsweep from 75 Hz to 40 Hz occurring in irregular sequences [c). All acoustic data were downsampled to 1 kHz to facilitate analysis. The 20-Hz call was previously analysed from these recordings and for another study [Acoustic recordings were analysed for two fin whale vocalizations: the 20-Hz call, a 1 s downsweep centred at 20 Hz  figure b and theer study  by usinger study . Based oer study ).Balaenoptera borealis) and blue whale  calls [Three months (representative of each season) with longer duty cycle recordings (1 h of continuous recordings) were manually inspected to identify song and non-song 20-Hz calls. Results showed that only 2.5% of the files contained non-song 20-Hz calls . Thus, we assumed that most 20-Hz calls analysed in this study were part of songs. Identification of 40-Hz calls using automatic detectors is challenging because of the frequency overlap with sei (s) calls . So, 40-s) calls , which cs) calls , as haviA call rate index was calculated as the total number of 20-Hz or 40-Hz calls detected in a week divided by the recording time, in hours, during that week, to reduce potential bias from the different duty cycles. Hereafter, we will in general refer to 20-Hz or 40-Hz call rates, but these strictly mean the corresponding call rate index. (b)https://www.mercator-ocean.fr/en/ocean-science/glorys/), produced with the ocean general circulation model NEMO (http://www.nemo-ocean.eu/), in an eddy-permitting configuration [http://www.science.oregonstate.edu/ocean). Outputs from these models were interpolated onto a weekly timescale and a spatial resolution of 0.25° × 0.25° to be used by the SEAPODYM-LTL model. The model predicts weekly mesozooplankton biomass on a global spatial grid (0.25° × 0.25°). Predictions for the period 1998–2019 are publicly available (https://marine.copernicus.eu/). The model validation is based on the climatological database COPEPOD that provides standardized mean zooplankton biomass values on a global spatial grid [Stable isotope analysis of skin and faeces indicates that fin whales from the study area feed primarily on zooplankton (mainly euphausiids and copepods) ,39. In aguration –43. Net ial grid ,43. (c)SEAPODYM-LTL estimates of mesozooplankton biomass (hereafter zooplankton biomass) were extracted for the weeks with acoustic recordings  and averaged across 0.25° × 0.25° grid cells centred around the hydrophone position. To determine the most appropriate spatial scale (i.e. the number of grid cells) for analysing SEAPODYM-LTL data in relation to acoustic data, the maximum detection range of 20-Hz and 40-Hz fin whale calls was estimated using the sonar equation :SNR=SL −el (RAM) ,46 was uFinally, a sensitivity test of the scale of data integration was performed by analysing annual and monthly patterns of estimates of zooplankton biomass at a range of scales. (d)Data from summer months (June to August) were excluded because the summer matches the end of fin whale migration through the Azores and whales are rare in the area ,47,48; hThe quasi-Poisson model  was used to describe the relationship between 20-Hz and 40-Hz call rates, and a set of independent variables: year, season  and zooplankton biomass. This modelling tool is especially suited to handle overdispersed count variables because it incorporates an overdispersion parameter that allows for more spread than the standard Poisson mean–variance relationship ,50. SepaF-test to account for overdispersion) were computed for the best model to validate each explanatory variable's statistical significance. The process started with the null model, and each explanatory variable was added sequentially until reaching the best model defined by the QAIC criterion. Moreover, the Wald test was applied to each parameter of the model to test the null hypothesis that the respective parameter is equal to zero.The best model was selected based on the lowest quasi-Akaike's information criterion (QAIC). Drop-in deviance tests Median detection ranges at the deployment locations were 64 km for the 20-Hz call and 18 km for the 40-Hz call . Therefore, SEAPODYM-LTL estimates of zooplankton biomass extracted for the weeks with acoustic recordings  were averaged across four grid cells of 0.25° × 0.25° centred around the hydrophone position (55 × 55 km). Changing the number of grid cells to nine (83 × 83 km) or 16 (194 × 194 km) had little or no effect on the annual and monthly patterns of estimated zooplankton biomass . (b)a). Conversely, 40-Hz call rates were low in autumn, increased in late winter, reached highest values in spring and decreased again in summer (b). Zooplankton biomass showed a clear peak in spring (April–May), decreased throughout the summer and early autumn and increased again in winter (c).Rates of the 20-Hz call increased in autumn, peaked in winter, decreased in spring and were null in summer a. Convern summer b. Zooplan winter c.Figure (c)a). 2012 had significantly lower call rates than all other years except 2009 . Overall, the model residuals did not show any pattern, indicating a good fit to the data. Most of the residuals were within the simulated envelope .Season was the most important predictor of the 20-Hz call, followed by year and one-week lagged call rates . Zooplankton biomass had no significant effect on the 20-Hz call a. 2012 h (d)c), the interaction between these two variables had no significant effect on 40-Hz call rates. Model residuals did not show outliers and indicated the model was adequate to describe the data .The best model for the 40-Hz call rate included only zooplankton biomass . Call rate increased with increasing zooplankton biomass c, the in. 4Our study shows that the production of 40-Hz calls in fin whales is positively associated with prey biomass, providing supporting evidence of a food-associated signal, as previously suggested ,26–29. T (a)-range communication [et al. [The winter peak in 20-Hz calls found in this study is consistent with the known seasonality of the fin whale song in the Northern Hemisphere ,16,35,56nication , supportnication ,22. Siminication ,22,58. C [et al. , male si [et al. . In this [et al. ,61. Thus [et al.  directed [et al. ,5. Howev [et al.  and furtc), the end of the singing season. It could also be argued that inter-annual variability in call rates results from differences in call detectability due to variations in background noise from shipping. Although noise levels in the study area did not vary significantly between 2010 and 2012 [The effect of year on singing activity was greatly influenced by 2012, which showed significantly lower call rates when compared to 2008, 2010 and 2011. The most plausible explanation is that decreased call rates in 2012 reflect lower numbers of fin whales in the area. This is partly supported by visual observations collected by the Fisheries Observer Program Data showing that fin whale encounter rates (sightings/100 km) were null in 2012, compared to 0.9 in 2010 and 9.2 in 2011. Year-to-year variability in fin whale numbers is usually attributed to environmental changes affecting prey distribution and abundance ,63. In tfigure 2and 2012 , effects (b)This study confirms the temporal separation between fin whale 40-Hz calls and 20-Hz calls . More imSome food-associated calls are produced only in feeding contexts, with animals adjusting call types or rates as a function of the type, quality or quantity of food available . More coOrcinus orca) [Petrochelidon pyrrhonota) feeding on insect swarms [Fin whales do not live in stable social groups  and the us orca) ) and mayus orca) . With thus orca) , there it swarms . Fin what swarms ,47. Fin t swarms . At presThe recent description of two acoustically tracked fin whales producing 40-Hz calls while moving past each other , gives s. 5Our study is the first to show a positive association between the production of the 40-Hz call and modelled biomass of prey, providing additional evidence of the use of this call in feeding contexts. Our findings are also consistent with earlier work indicating that the song-forming 20-Hz call is used in reproductive contexts, but the absence of a relationship with prey biomass does not support the assumption that this call is used by males to advertise a food source and attract potential mates. Instead, the 20-Hz song may be a male acoustic display used in intersexual and intrasexual interactions. Our study also illustrates how spatio-temporally resolved simulations of zooplankton biomass, which is challenging to measure in the field, can provide valuable insights into the environmental context and potential functions of baleen whale vocalizations.Understanding call function and monitoring vocal behaviours associated with the state of individuals or groups , habitat quality (e.g. food resources) or animal density  can help identify functional habitats, predict negative human impacts and support conservation planning . InformaClick here for additional data file.


The effort, focus, and time to collect data and train EMG pattern recognition systems is one of the largest barriers to their widespread adoption in commercial applications. In addition to multiple repetitions of motions, including exemplars of confounding factors during the training protocol has been shown to be critical for robust machine learning models. This added training burden is prohibitive for most regular use cases, so cross-user models have been proposed that could leverage inter-repetition variability supplied by other users. Existing cross-user models have not yet achieved performance levels sufficient for commercialization and require users to closely adhere to a training protocol that is impractical without expert guidance. In this work, we extend a previously reported adaptive domain adversarial neural network (ADANN) to a cross-subject framework that requires very little training data from the end-user. We compare its performance to single-repetition within-user training and the previous state-of-the-art cross-subject technique, canonical correlation analysis (CCA). ADANN significantly outperformed CCA for both intact-limb (86.8–96.2%) and amputee (64.1–84.2%) populations. Moreover, the ADANN adaptation computation time was substantially lower than the time otherwise devoted to conducting a full within-subject training protocol. This study shows that cross-user models, enabled by deep-learned adaptations, may be a viable option for improved generalized pattern recognition-based myoelectric control. Electromyography (EMG) pattern recognition has been pursued as a way to control prosthetic devices for decades; however, there is a growing interest in its use for a wider range of commercial applications  have recently been proposed as a powerful subject independent model  were recorded each at 1,000 Hz using a 16-bit analog-to-digital converter. Signals were preprocessed using an analog 5th-order anti-aliasing filter with cut-off frequency of 500 Hz. For intact-limb subjects, electrodes were placed around the circumference of the forearm at the widest area with positions being as similar as possible across subjects. A similar approach was taken with amputee subjects, although electrode positions deviated slightly to best cover the residual muscle. Data were further processed using digital filters to minimize powerline interference (60 Hz notch filter) and motion artifacts (20 Hz high-pass filter). Finally, overlapping windows were extracted with window size and window increment of 151 and 50 ms, respectively.xi is the ith d-dimensional sample of the expert subject dataset, and ti is the ith k-dimensional sample of the target subject dataset. Within our study, k and d are the same since CCA always was performed between pairs of subjects using the same feature set. Features within the datasets are standardized to zero-mean and unit-variance to ensure that the dataset is centered and avoid the need to learn a bias term. The datasets are considered paired by organizing the datasets to have an identical progression of class labels. In modern implementations of CCA .CCA is a statistical method appropriate for domain adaptation because its objective is to maximize correlation between two paired datasets: W through an additional term λwj, where λ is a small positive scalar (0.04 in this study) and wj is the jth eigenvector of W.Regularization is introduced in Equation (2) to penalize large parameter values of After the linear projection has converged to a solution, the data from a test user can be mapped to the space of the expert subject using Equation (3).Following the convention proposed by Khushaba , CCA wasFive handcrafted feature sets were chosen to undergo CCA projection in this analysis due to their prevalence across the literature:The Hudgins' time domain (TD) features are perhaps the most commonly used EMG feature set and include the mean absolute value, zero crossings, slope sign change, and waveform length features  feature sets have been proposed as a robust alternative when data are acquired at 200 Hz , although subsequent studies have demonstrated strong performance at 1,000 Hz as well. The LSF4 feature set contains maximum fractal length, l-score, mean-squared ratio, and Willison's amplitude  classifier was adopted to classify these handcrafted feature sets due to its extensive use in EMG pattern recognition literature and low computational complexity  dimensional input vectors. Within each convolutional block, the inputs were transformed by a convolutional layer that consisted of 64 kernels of size 1 × 21 (single channel × 21 samples). This produced 64 feature maps of activation values. Afterwards, these activations were regularized by batch normalization to stabilize and accelerate convergence during model training. The batch normalization transform is given in Equation (4), where xk). This was transformed to a 7 × 1 output vector which represented the number of classes in the dataset. A softmax was applied to the output and the highest activated neuron was assigned as the predicted gesture.The model hyper-parameters were selected according to values found suitable in past experiments and through iterative optimization. The learning rate was 0.04047 and the Adam optimizer was employed   and a testing set (eight repetitions) for each subject  were used as the repetitions within the analysis. The two independent variables under investigation were the population type  and the gesture recognition pipeline . If a significant difference was found at the whole study level, subsequent one-way RMANOVA tests would be conducted on these significant variables. Finally, if the one-way RMANOVA tests were significant, post-hoc t-tests would be conducted using a Bonferroni correction.Statistical significance was evaluated using a two-way repeated-measures analysis of variance (RMANOVA) test with a significance value of The performance of the within-subject, single-repetition, and cross-subject models for the intact-limb and amputee datasets are given in p = 0.007), gesture recognition pipeline (p~0), and their interaction (p~0) on gesture recognition accuracy. The one-way RMANOVA investigating the effect of population found a significant effect for all pipelines except TD, TDAR, TDPSD, LSF4, and LSF9 in the cross-subject evaluation framework .Prior to conducting the RMANOVA, the assumptions of normality and sphericity were verified to ensure this parametric test was appropriate. The two-way RMANOVA analysis revealed a significant effect of population (p~0). Post-hoc tests revealed ADANN performed significantly better for intact-limb subjects than all other pipelines across evaluation frameworks (p < 0.05), except the within-subject framework TDAR, TDPSD, LSF4, LSF9, and CNN pipelines . For reference, the accuracies of the intact-limb subjects using the ADANN pipeline and within-subject TDAR, TDPSD, LSF4, LSF9, and CNN pipelines were 96.2 ± 2.8, 93.9 ± 3.1, 95.4 ± 3.1, 95.8 ± 2.7, 96.1 ± 2.5, and 97.4 ± 1.5, respectively. This indicated that despite being evaluated under the cross-subject condition, ADANN achieved an accuracy competitive with pipelines trained with full training protocols. Moreover, the ADANN pipeline resulted in consistently higher accuracies for each gesture when compared against all other intact-limb single-repetition and cross-user pipelines.The one-way RMANOVA investigating the effect of pipeline was found to be significant for intact-limb subjects . This indicated that the single-repetition used for adaptation in the cross-user CCA frameworks would be leveraged better by independently training a model for this dataset.The prior state of the art cross-subject technique, CCA, also achieved reasonable cross-subject performance for intact-limb subjects. In particular, CCAp~0). The ADANN pipeline was likewise found to significantly outperform most pipelines, with the exception of the within-subject framework TDPSD, LSF4, LSF9, and CNN . For reference, the accuracies of amputee subjects using the ADANN pipeline and within-subject TDPSD, LSF4, LSF9, and CNN pipelines were 84.1 ± 5.3, 82.4 ± 5.5, 83.9 ± 5.6, 84.1 ± 5.3, and 84.3 ± 5.5, respectively. Similar to the intact-limb results, the ADANN pipeline accuracy for amputee subjects was consistently higher for each gesture when compared against the other single-repetition and cross-user pipelines.The one-way RMANOVA investigating the effect of pipeline was also found to be significant for amputee subjects  and the within-subject model , and presumably achieve at least similar performance. This work did not consider the confounding factors typically encountered alongside real world applications of myoelectric control . Consequently, future works should consider the combination of reducing training burden using ADANN while also incorporating these dynamic factors.A visual summary of the results found in this manuscript are given in escheme@unb.ca).Publicly available datasets were analyzed in this study. This data is available upon request to Erik Scheme (The studies involving human participants were reviewed and approved by University of New Brunswick Research Ethics Board. The patients/participants provided their written informed consent to participate in this study.Access to the datasets were provided by ES. The experimental design was planned by EC, AP, and ES. The experiment and statistical analysis were carried out by EC. The manuscript was written by EC, with edits provided by AP and ES. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Polyamines (PAs) are polycationic biomolecules containing multiple amino groups. Patients with HIV-associated neurocognitive disorder (HAND) have high concentrations of the polyamine N-acetylated spermine in their brain and cerebral spinal fluid (CSF) and have increased PA release from astrocytes. These effects are due to the exposure to HIV-Tat. In healthy adult brain, PAs are accumulated but not synthesized in astrocytes, suggesting that PAs must enter astrocytes to be N-acetylated and released. Therefore, we tested if Cx43 hemichannels (Cx43-HCs) are pathways for PA flux in control and HIV-Tat-treated astrocytes. We used biotinylated spermine (b-SPM) to examine polyamine uptake. We found that control astrocytes and those treated with siRNA-Cx43 took up b-SPM, similarly suggesting that PA uptake is via a transporter/channel other than Cx43-HCs. Surprisingly, astrocytes pretreated with both HIV-Tat and siRNA-Cx43 showed increased accumulation of b-SPM. Using a novel polyamine transport inhibitor (PTI), trimer 44NMe, we blocked b-SPM uptake, showing that PA uptake is via a PTI-sensitive transport mechanism such as organic cation transporter. Our data suggest that Cx43 HCs are not a major pathway for b-SPM uptake in the condition of normal extracellular calcium concentration but may be involved in the release of PAs to the extracellular space during viral infection. The human immunodeficiency virus (HIV), identified in 1980, is a lentivirus that attacks the immune system and brain of humans and other primate species  and is rSince combined highly active antiretroviral therapies (HAART) became the standard treatment for HIV, life expectancy has increased . Neverth2+ from its binding site, resulting in NMDA receptor activation and increasing its capacity to allow calcium ion flux [HIV-Tat is a protein made by the HIV virus that can promote efficient transcription and replication of the HIV genome ,11. Moreion flux ,13. HIV-ion flux ,15. Expoion flux .1-acetyl-SPM. Taking into account that: (i) glial cells lack the biosynthetic enzymes for PA synthesis [A key finding by Merali and co-authors 2014) showed that astrocytes are the principal cells in which HIV-1 Tat protein activates the enzyme spermidine/spermine acetyltransferase (SSAT) that converts the polyamine (PA) spermine (SPM) to Nynthesis ,18 but aynthesis ,20,21, a14 showedynthesis ,23,24, wynthesis ,26. Takeynthesis . Therefoynthesis ,29 and Aynthesis . The relynthesis  and nervynthesis . Ultimatynthesis ,33. Sincynthesis  and PAs ynthesis ,36, gliaSince PA uptake, N-acetylation, and release are key aspects of HAND, it is important to understand how PAs enter cells. On the basis of previous publications on PA permeability via large pores such as connexin-43 (Cx43) ,38,39 anThe purpose of the present study was to determine if Cx43 HCs and/or OCTs are major transmembrane proteins involved in PA uptake in astrocytes to replenish the pool of PA for their N-acetylation in patients with HAND. To study the potential pathways for PA uptake in astrocytes expressing HIV-Tat protein, we incubated cultured astrocytes with SPM tagged with biotin  a2+, Mg2+), PAs enter the astrocytes via a PTI-sensitive pathway, but not via Cx43 HCs. Interestingly, HIV-Tat protein increases the b-SPM uptake/accumulation in Cx43 siRNA-treated astrocytes, but not in mock-transfected astrocytes and siRNA Cx43-treated cells without HIV-Tat. This suggests the possibility that Cx43 HCs act as a release pathway for PAs in the presence of HIV-Tat. Taken together, these data suggest that Cx43 is not an uptake pathway for SPM when divalent cations are in the steady state range but may be involved in the release of PAs to the extracellular space, particularly during viral infection.Our results indicate that in normal millimolar concentrations of divalent cations  mice obtained from Charles River Laboratories .Primary cultures of astrocytes were prepared from the neocortex of 1–3-day-old C57 mice, as previously described . Briefly® West Dura Extended Duration Substrate; Rockford, IL, USA) as described by the manufacturer, and the signal was quantified using a gel documentation system . The image was obtained using the Image Lab software .After 4 weeks in culture, cortical astrocytes were harvested, pelleted, and resuspended in homogenization buffer as previously described . BrieflyAfter 4 weeks in culture, astrocytes were trypsinized and plated 50,000 cells per well in a 24 well plate in which cover glasses were already placed in each well. Immediately after plating, cells were transfected with 20 nM of siRNA targeting Cx43  and 1.2 µL of HiPerFECT  in 500 µL of cell culture media , according to the manufacturer’s instructions. Transfected astrocytes were used for experiments after 72 h. Prior to the experiments, we determined the optimal time and concentration of siRNA necessary to obtain the maximal decrease in Cx43 expression (data not shown).2HPO4·7H2O, and 2.2 NaH2PO4, pH 7.4. Cells were treated at the time of transfection with 50 ng/mL of HIV-Tat 72 h prior to the experiment.HIV-1 IIIB Tat (HIV-Tat) Recombinant Protein  was obtained from the NIH AIDS Reagent Program . The protein was reconstituted in phosphate-buffered saline (CPBS), which contained in mM: 133 NaCl, 2.7 KCl, 8.2 NaThe trimer44NMe is a novel polyamine transport inhibitor (PTI) ,44,45 thBiotinylated spermine (b-SPM) was synthesized following established methods to conjugate N-hydroxy-succinimide esters of haptens with amino group-containing molecules ,47. In s2 2; CaCl2 2.5; Glucose 10). We added 500 µL of the saline solution with 80 µM of b-SPM and incubated the mixture for 1 h at 37 °C with gentle shaking. Experimental groups receiving HIV-Tat were incubated with HIV-Tat protein at the same time as the transfection (72 h prior to the experiment). Experimental groups receiving PTI were treated one hour prior to b-SPM treatment and also included with b-SPM for the one-hour incubation period. After the incubation period ended, the solution was removed, and the cells were rinsed 1 time with 500 µL of cold 4% paraformaldehyde (PFA) in phosphate-buffered saline solution . The PFA was exchanged with 1 mL of fresh cold PFA, and cells were incubated at 4 °C for 1 h. Fixation is very important to maintain the fine structure of the tissue, but even more to chemically link the PAs to adjacent proteins. The cells were fixed without a previous washing step, otherwise the PAs would be lost during washing steps. After incubation, the cells were washed 3 times with PBS or with 0.3% Triton X (triton) in PBS at room temperature for 5 min. Cells were then incubated in 500 µL/well of Rhodamine Avidin D  solution (1/200 dilution in PBS) at room temp for 2 h, after which, the cells were washed with PBS for 10 min, 20 min, 30 min, and 1 h at room temperature. These washing steps were critical to decrease non-specific fluorescence due to residual Rhodamine Avidin D that was not attached to the b-SPM. Cell nuclei were stained using DAPI (1/5000 in PBS) for 10 min and subsequently washed 3 times with PBS for 5 min to remove residual DAPI. The cover glasses were carefully removed from the 24 well plate and adhered to microscope slide using Vectashield mounting media ; then, they were dried for 24 h prior to their examination using confocal microscopy.For the uptake experiments, we removed the media from the transfected cells and washed them with saline solution at 37 °C  . Six images per slide were obtained by LSCM using Rhodamine Red-X (580 nm) and DAPI (455 nm) lasers and a 60× objective. Laser exposure intensity was the same for all images in a given experiment. Initially, we set the laser exposure intensity using positive control cells and then we switched to a negative control slide to reduce laser exposure to the minimum. Third, we switched back to the positive control slide to see the b-SPM signal and make any last and minor adjustments to the laser intensity before proceeding to capture images from all slides without changing any parameters. Images were analyzed using ImageJ  and Microsoft Excel . We used the following formula Equation (1) to calculate the cell fluorescence in all the images, taking into account differences in the numbers of cells per image .(1)Cellt-test . Data from the b-SPM experiments were analyzed using a two-way ANOVA, Tukey’s multiple comparison, and Sidak’s multiple comparison tests . A one-way ANOVA, Tukey’s multiple comparison, and Dunnett’s multiple comparison tests were used to analyze data in Figure 5. A value of p < 0.05 was considered significant.All statistical analyses were performed using GraphPad Prism Version 8.4.3 (471) . Data from Western blots were analyzed using an unpaired Student’s n = 3/group). Although we only show this one example of Cx43 knock-down, Western blot was used to assess knock-down in all siRNA experiments, and it was equally effective in all (data not shown).To assess the efficacy of the siRNA treatment in each experiment, we used Western blot to measure the expression of Cx43 in mock-transfected control astrocytes (HiPerFect with no siRNA) versus those transfected with siRNA targeting Cx43. The samples for Western blot were run in parallel with the astrocytes used for b-SPM uptake. As shown in In the first series of experiments, we determined the contribution of Cx43 hemichannels to b-SPM uptake into cultured astrocytes. Visualization of 80 µM b-SPM uptake depends upon the high affinity between avidin and biotin ,50, withFor the experimental groups, we measured cell fluorescence as a semi-quantitative measure of b-SPM uptake into control astrocytes (DMEM), mock-transfected astrocytes (DMEM + 1.2 µL of HiPerFECT), and those transfected with siRNA targeting Cx43 (DMEM + 20 nM of siRNA targeting Cx43 + 1.2 µL of HiPerFECT). We found significant uptake of b-SPM into astrocytes of all three experimental triton groups when compared with the no triton control and the no b-SPM groups. The level of uptake was similar for all three treatments including astrocytes in which Cx43 was knocked down, which suggests that Cx43 is not a major pathway for SPM uptake under these conditions .In the next series of experiments, the effect of HIV-Tat protein on uptake/accumulation of b-SPM in astrocytes was examined. We noted that prior work in cancer cells by Mani et al. revealed that HIV-Tat was a potent inhibitor of PA uptake , but in On the basis of the findings of experiments 1 and 2 described above, we found that Cx43 hemichannels did not appear to be a major pathway for PA uptake into astrocytes when the extracellular solution contained normal levels of divalent cations. We have previously shown that trimer44NMe  can inhibit astrocyte cell proliferation in response to exogenously applied spermidine when de novo synthesis of PAs is blocked . We hereThe next experiment  was desiAlthough PA levels in brain have been associated with the severity of aging, dementia, neurological syndromes, and HIV-associated neurocognitive disorders (HAND), little is known about PA flux in the brain ,53. IndeWe found that astrocytes import exogenously applied b-SPM in control conditions, as seen by an increase in cell fluorescence upon incubation with rho-avidin. This uptake was blocked by PTI, but not by knockdown of Cx43 hemichannels. The Trimer44NMe PTI is a specific blocker for PA transport, which is a competitive inhibitor of PA uptake ,55. We h2+ and 2.0 mM Mg2+). However, it is well established that Cx43 HCs are predominantly closed when there are physiological levels of these extracellular divalent cations [2+ [Furthermore, our results suggest that Cx43 HCs are not the main pathway for b-SPM uptake in primary-cultured cortical astrocytes. In the present experiment, we applied exogenous b-SPM to astrocyte cultures in the presence of physiologically high concentrations of divalent cations   and incrIn the current study, astrocytes were treated with HIV-Tat protein for 72 h prior to incubation with b-SPM. We found that astrocytes took up b-SPM, but there was no difference between the uptake/accumulation of b-SPM between control astrocytes and astrocytes treated with HIV-Tat. This finding is consistent with the study of Merali et al. (2014), which demonstrated that PA levels in HIV-Tat-treated astrocytes were stable. Surprisingly, when we treated astrocytes with HIV-Tat protein and knocked-down Cx43 in these cells, there was a significant increase in cell fluorescence when compared to HIV-Tat alone, indicating that uptake/accumulation of b-SPM was increased. Furthermore, PTI completely blocked b-SPM uptake/accumulation even in HIV-Tat-treated astrocytes in which Cx43 was knocked down.In the present study, we examined the steady state levels of PAs at 1 h after uptake. The data obtained do not provide information about b-SPM that could have been taken up into astrocytes and subsequently released, which would have been consistent with the increased PA flux described by Merali et al. (2014). Furthermore, we measured cell fluorescence which is due to the attachment of rho-avidin to the biotin molecule that was tagged to SPM. If the b-SPM molecule is transformed to b-Ac-SPM due to activation of spermine/spermidine acetyl transferase, one would expect to still see an increase in cell fluorescence due to an interaction between rho-avidin and the biotin which is now tagged to Ac-SPM.Taking into consideration the findings of Merali (2014) and the data obtained in our experiments, we propose the following model . In contThe actions of the PAs, SPM, and SPD on neuronal receptors and channels has been well studied ,38,39,40In conclusion, our data indicate that PAs enter astrocytes via a PTI-sensitive pathway, and Cx43 is not the main pathway for PA uptake in astrocytes. Import of PAs can be blocked by PTI with and without the presence of HIV-Tat protein. This suggests that b-SPM uptake may be via organic cation transporters or another major transmembrane protein that has not been identified yet . Interes


In the definitive study, these 20 miRNAs were analyzed in blood samples of healthy dogs (n = 6), dogs with PDH  and dogs with a csACT . In dogs with PDH, six miRNAs  were differentially expressed compared to healthy dogs. Of one miRNA, miR-122-5p, the expression levels did not overlap between healthy dogs and dogs with PDH (p = 2.9x10−4), significantly decreased after hypophysectomy (p = 0.013), and were significantly higher (p = 0.017) in dogs with recurrence (n = 3) than in dogs without recurrence for at least one year after hypophysectomy (n = 7). In dogs with csACTs, two miRNAs (miR-483-3p and miR-223-3p) were differentially expressed compared to healthy dogs. Additionally, miR-141-3p was expressed significantly lower (p = 0.009) in dogs with csACTs that had a histopathological Utrecht score of ≥ 11 compared to those with a score of <11. These results indicate that circulating miRNAs have the potential to be non-invasive biomarkers in dogs with Cushing's syndrome that may contribute to clinical decision making.Canine Cushing's syndrome (hypercortisolism) can be caused by a pituitary tumor  or a cortisol-secreting adrenocortical tumor (csACT). For both cases, non-invasive biomarkers that could pre-operatively predict the risk of recurrence after surgery would greatly impact clinical decision making. The aim of this study was to determine whether circulating microRNAs (miRNAs) can be used as diagnostic (presence of PDH or csACT) and/or prognostic  non-invasive biomarkers for canine Cushing's syndrome. After a pilot study with 40 miRNAs in blood samples of healthy dogs ( Spontaneous Cushing's syndrome, or hypercortisolism, is one of the most commonly diagnosed endocrinopathies in dogs . It is cPTTG1) was previously reported to be associated with disease-free interval after surgery, but can only be assessed post-operatively  or an adrenocortical carcinoma (ACC) , but makHaving non-invasive biomarkers that can pre-operatively predict the risk of recurrence after surgery would greatly impact clinical decision making. Potential candidates for such non-invasive biomarkers are circulating microRNAs . MiRNAs are single-stranded, non-coding RNAs of ~20–24 nucleotides in size . They fuThe aim of this study was to determine whether circulating miRNAs can be used as non-invasive biomarkers for canine Cushing's syndrome. We hypothesized that we could identify circulating miRNAs of which the expression profiles are associated with the presence or absence of PDH or a csACT, the size of the pituitary tumor and whether PDH recurred after hypophysectomy, and the histopathological assessment of a csACT.n = 3), dogs with csACTs (n = 4), and dogs with PDH (n = 3). Written informed consent was obtained from the owners for the participation of their animals in this study. Serum samples were available from the healthy dogs and dogs with csACTs, while EDTA plasma samples were available from the dogs with PDH. After sample collection, serum or EDTA plasma was obtained by centrifuging whole blood for 5 min at 4,000 rpm. The serum and EDTA plasma samples were stored at −70°C until use.For the pilot study, we included blood samples of healthy dogs  combined with high-dose dexamethasone suppression test), or non-suppressible hypercortisolism combined with findings of symmetric bilateral adrenal enlargement  and the absence of unilateral adrenal enlargement (indicating an ACT) on ultrasonography or CT, together with findings of pituitary enlargement on CT or MRI. The diagnosis of a csACT was made by non-suppressible hypercortisolism demonstrated with endocrine testing, combined with the presence of an ACT found on ultrasonography or CT. All pituitary tumors were classified as adenomas based on histopathological assessment and absence of detectable metastases . HistopaMiRNA was isolated using the miRNeasy Serum/Plasma Kit , approved for animal and human plasma and serum samples, according to the manufacturer's instructions. The serum and EDTA plasma samples were thawed on ice. As sample input 150 μL was used, and volumes of the other components were adjusted accordingly. The miRNeasy serum/plasma kit was combined with the RNA Spike-In Kit, For RT  for miRNA isolation quality control. The mixture of spike-ins UniSp2, UniSp4 and UniSp5 was prepared according to the manufacturer's instructions, and 1 μL of the spike-in mixture was added per sample. After isolation, the miRNA eluates were stored at −20°C until use. For subsequent cDNA synthesis, miRNA samples were thawed on ice. The cDNA was synthesized using the miRCURY LNA RT Kit  according to the manufacturer's instructions, using 2 μL of RNA template per 10 μL total reaction volume. For cDNA synthesis quality control, 0.5 μL of a mixture of spike ins UniSp6 and cel-miR-39-3p (Qiagen) was added to the cDNA reaction. The cDNA samples were stored at 4°C when real-time quantitative polymerase chain reaction (RT-qPCR) was performed within four days after cDNA synthesis, or at −20°C when the RT-qPCR was performed at a later timepoint.For all samples, a quality control was performed to detect the presence of spike-ins UniSp2, UniSp4 and UniSp5 (to assess efficiency of miRNA isolation), of UniSp6 and cel-miR-39-3p (to assess efficiency of cDNA synthesis), and of the miRNAs miR-23a and miR-191 (to assess efficiency of endogenous miRNAs detection). To obtain miRNA-specific primers we checked the canine sequences on the miRBase database , and ord−ΔΔCT method , UniSp6 (cDNA spike-in control), one non-template control, five potential reference miRNAs, and 40 potential target miRNAs . DetectT method .n = 6), pre-operative blood samples of dogs with csACTs (n = 26), and both pre- and post-operative samples of dogs with PDH (n = 19). The blood samples used in the pilot study were also included but reanalyzed in the definitive study. For the healthy dogs both EDTA plasma and serum samples were available. For the dogs with PDH only EDTA plasma samples were available, while for the dogs with csACTs only serum samples were available. Because of these differences in sample types, miRNA expression levels were not compared between dogs with PDH and with csACTs, but only between dogs with either PDH or csACTs and healthy controls of corresponding sample types. Written informed consent was obtained from the owners for the participation of their animals in this study. For all dogs with PDH, surgery and sample collection were performed at the Faculty of Veterinary Medicine in Utrecht, as was the sample collection for the healthy dogs. For dogs with csACTs, the surgery and sample collection were performed at the Utrecht University Clinic or at external clinics. In case of external clinics, the samples were sent by post to the Faculty of Veterinary Medicine in Utrecht. Samples for the dogs with PDH and csACTs were collected between 2015 and 2020, samples for the healthy dogs were collected between 2019 and 2020. The clinical data of the dogs included in this study can be found in an online data repository .To determine the pairwise variance and stability of miRNA expression, the geNorm  method fT method . Normal The datasets generated for this study can be found in the DataverseNL data repository .The quality control results [available through online data repository ] indicatOf the 40 target miRNAs assessed in the pilot study [results available through online data repository ], 6 weren = 44; 6 healthy dogs and 19 dogs with PDH, pre-op and post-op), in serum samples , and in the combined EDTA plasma/serum samples. The combination of miRNAs was interpreted as suitably stable when the Pairwise Variance (V-score) was lower than 0.15 (V0.15)  . However5) . The lowest V-score  in the combined EDTA plasma/serum samples group (“All”) was achieved with a combination of 12 miRNAs  was used as normalization control.For the subsequent analyses, we analyzed the expression levels of all 20 miRNAs. The geometric mean of the Vn = 19) compared to healthy dogs (n = 6): miR-122-5p , miR-141-3p (p = 0.028), miR-222-3p (p = 0.008), miR-375-3p (p = 0.001), and miR-483-3p (p = 0.009) (p = 0.036)  . One miR= 0.036) .p = 0.013) and of miR-141-3p (p = 0.035) significantly decreased after surgery . The expression levels of miR-126-5p and miR-483-3p did not change in the post-op samples .Of all 19 dogs with PDH, post-op samples were available. The timepoint of post-op sample collection was at median 3 days after hypophysectomy (range 1–7 days). The six differentially expressed miRNAs were analyzed in the post-op EDTA plasma samples, to determine whether their expression levels either decreased or increased after surgery. Paired-sample analyses of these six miRNAs showed that the expression levels of miR-122-5p  were correlated with the P/B value. None of the six differentially expressed miRNAs were significantly correlated with the P/B value, nor were any of the other fourteen miRNAs .p = 0.017) and miR-222-3p (p = 0.030) were expressed significantly higher in the dogs with recurrence than in the dogs without recurrence , 7 dogs had no recurrence of hypercortisolism and had follow-up of at least one year after surgery , and 4 dogs had no recurrence of hypercortisolism but follow-up times of less than one year after surgery . The 3 dogs with reported recurrence were subsequently included in the “Recurrence” group, while the 7 dogs without recurrence and with follow-up time of at least one year after surgery were included in the “No recurrence” group. In assessing the expression profiles of the six differentially expressed miRNAs in the pre-operative samples, we found that miR-122-5p (currence .p = 0.105), the other five miRNAs were still significantly higher in dogs with PDH compared to healthy dogs (p = 2.9x10−4) because still no overlap between sample groups was observed, but the p-values of the other four miRNAs increased .Because the clinical application of miRNAs is limited when 12 miRNAs need to be assessed for data normalization, we wanted to determine whether the detected differences could still be observed when only one reference miRNA was used. As the most stably expressed miRNA in all sample groups, we chose to use the expression of miR-191-5p for data normalization. Although miR-126-5p lost its significance (thy dogs  when norp = 0.017) and miR-222-3p (p = 0.017) were still significantly higher in the recurrence group  compared to healthy dogs (n = 6): miR-483-3p (p = 0.020), while one miRNA was expressed significantly lower in dogs with a csACT: miR-223-3p (p = 0.038)  .n = 26) was 8.2 % (range 0.3–50.7 %), and the median Utrecht score was 12.6 (range 4.3 – 57.7). Although miR-132-3p and miR-141-3p showed a moderate correlation with the Utrecht score, either positively (miR-132-3p) or negatively (miR-141-3p), these correlations were not significant (The median Ki67 PI in the csACTs (nificant . None ofnificant .n = 9) or ≥ 11 (n = 17). When comparing miRNA expression between these groups, miR-141-3p was significantly higher in in the group with an Utrecht score of <11 than in the group with an Utrecht score of ≥ 11   , 34–36. g cancer , pancreag cancer  and hepag cancer , it is ag cancer . The tumg cancer , and couAlthough miR-122 expression has a clear link with cancer, there are also other diseases in which miR-122 expression can be dysregulated. Because miR-122 is derived from hepatocytes, it was found to be overexpressed in serum of dogs with different types of liver diseases , 30, 40.Another miRNA that was overexpressed in dogs with PDH compared to healthy dogs, as well as in dogs with PDH that had recurrence after hypophysectomy compared to dogs that did not have recurrence, is miR-222-3p. MiR-222 is overexpressed in many different human cancer types . A recenOf the other four significantly altered miRNAs in PDH found in this study, only one was expressed significantly lower compared to healthy controls: miR-126-5p. MiR-126 has also been reported to be downregulated in other diseases in humans, including in diabetes mellitus . In contIn dogs with csACTs, miR-483-3p was significantly overexpressed compared to healthy dogs. In humans, miR-483-5p, the reverse miRNA from the miR-483 locus, is one of the most commonly reported circulating miRNA that is overexpressed in patients with ACC compared to those with ACA –53. Althin vitro and in vivo  or preventive health screenings . Written informed consent was obtained from the owners for the participation of their animals in this study.KS and SG conceived and designed the experiments. KS, AV, AS, and ET-S performed the experiments. KS, AV, HK, FR, BM, and SG analyzed the data. HK, SD, FF, SN, and BM contributed materials. KS, AV, and SG wrote the paper. All authors contributed to the article and approved the final version of the manuscript for submission.We thank the Utrecht University Open Access Fund for partly reimbursing the Article Processing Fee.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Galanin (GAL) is a nociceptive transmitter or modulator in the trigeminal sensory system. In this study, GAL expression was investigated in the rat dura mater to demonstrate its possible function in headache using immunohistochemical techniques. The cerebral falx and cerebellar dura mater received abundant blood and nerve supply, and were significantly thicker compared to other portions in the cerebral dura mater. GAL-immunoreactivity was expressed by cell and nerve fiber profiles. Presumed macrophages and dendritic cells contained GAL-immunoreactivity, and co-expressed with CD11b-immunoreactivity. Many isolated and perivascular nerve fibers also showed GAL-immunoreactivity. In addition, GAL-immunoreactive nerve fibers were present in the vicinity of macrophages and dendritic cells with either GAL- or ED1-immunoreactivity. GAL-immunoreactive cells and nerve fibers were common in the cerebral falx and cerebellar dura mater and infrequent in other portions. And, GAL-immunoreactive nerve fibers usually co-expressed calcitonin gene-related peptide (CGRP)-immunoreactivity. In the trigeminal ganglion, a substantial proportion of sensory neurons innervating the dura mater contained GAL-immunoreactivity , and co-expressed CGRP-immunoreactivity (2.7 ± 2.1%). The present study may suggest that GAL is associated with nociceptive transduction or modulation in the dura mater. GAL also possibly plays a role in the immune mechanism of the dura mater. The dura mater and arachnoid mater keep the cerebrospinal fluid within the cranial cavity. The dural sinuses are supported by the cranial dura mater and send blood from the brain to the heart through the internal jugular vein2. The blood and nerve supply of the meninges is abundant in the dura mater but not in the arachnoid mater or pia mater3. In addition, the dura mater contains many macrophages and dendritic cells5. These cells may be able to protect the brain against foreign invaders and damages.The cranial dura mater is a hard membrane that surrounds the brain with the arachnoid mater and pia mater8. These neurons contain calcitonin gene-related peptide (CGRP), a putative nociceptive transmitter11. CGRP-containing TG neurons supply the dura mater with free nerve endings and perivascular endings12. In the trigeminal system, CGRP causes neurogenic inflammation13 such as vasodilation and plasma extravasation14. A previous in vitro study using the RAW264.7 cell line has demonstrated that CGRP can also regulate the synthesis of cytokines in macrophages15. Thus, CGRP-containing TG neurons may contribute to nociceptive transduction and immune response in the dura mater.The dura mater is innervated from the trigeminal ganglion (TG). Previous retrograde tracing studies have demonstrated that sensory neurons innervating the dura mater have small to medium-sized cell bodies in the TG19. This peptide can modulate or inhibit action potentials in brain and spinal cord neurons. In the sensory ganglion, GAL is mainly expressed by small neurons22. Previous double immunofluorescence studies have also demonstrated the co-expression of GAL and CGRP in the TG24. Similar to CGRP-containing TG neurons, GAL-containing TG neurons transduce nociceptive information from cranial structures to the brainstem25. Our previous study also demonstrated that GAL and its receptors in the TG respond to nerve transection and suggested that they protect TG neurons from damage through their autocrine and paracrine mechanisms22. In addition, GAL is expressed by macrophages derived from peripheral blood mononuclear cells and is shown to affect their activity by modulating some chemokines and cytokines27. Thus, we hypothesize that GAL in the dura mater functions in the nociceptive transmission and defense mechanism by the immune system.Galanin (GAL), another member of the neuropeptide family, is distributed in the central and peripheral nervous systemsDetermining the distribution and origin of GAL and its relationship with immune cells in the dura mater may facilitate our understanding of GAL function in the nociceptive and immune defense mechanisms. However, little is known about the distribution or function of GAL in the dura mater. In the present study, the distribution of GAL was examined in the rat dura mater. The relationship between GAL- and CGRP-containing profiles was also investigated by a double immunofluorescence method. In addition, a retrograde tracing method was performed to determine the origin of GAL-containing nerve fibers in the dura mater.28. The outer layer consisted of thin fibrous tissues containing simple squamous cells. Meanwhile, the inner layer of the dura mater was of various thicknesses  were larger than round type cells . Both types of GAL-ir cells were usually distributed in the deep layer of the dura mater. They were also abundant in the cerebral falx and cerebellar dura mater, and around the olfactory bulb -positive neurons were present in the rostral 2/3 of the TG. A substantial proportion  of FG-positive TG neurons were GAL-ir. Moreover, half  of FG-positive TG neurons showed CGRP-immunoreactivity . Thirty-one % (80/262) and 48.9% (128/262) of total FG-positive neurons had small (< 600 µm2) and medium-sized cell bodies (600–1200 µm2), respectively. Large FG-positive neurons were relatively infrequent in the TG . FG-positive TG neurons with small to medium-sized cell bodies contained GAL-  and CGRP-immunoreactivity . All FG-positive neurons which co-expressed GAL- and CGRP-immunoreactivity had small cell bodies Twelve male Wistar rats  were used in this study. They were purchased from CLEA Japan, Inc. (Japan). Eight and 4 rats were used for tissue preparation and retrograde tracing study, respectively.41. All experiments were performed after being reviewed and approved by Institutional Laboratory Animal Care and Use Committee of Tohoku University (2020DnA-040).All animal experiments were performed according to The Guidelines for Care and Use of Laboratory Animals in Tohoku University, and the Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6), which are in line with ARRIVE guidelines42. The brain with the meninges was dissected and immersed in the same fixative. Then, the samples were immersed overnight in a phosphate-buffered saline containing 20% sucrose and frozen with dry ice. The whole brain was frontally and serially sectioned at 8 µm and thaw-mounted on silane-coated glass slides (n = 4). The caudal 1/3 portion of the brain, including the brainstem and cerebellum, was also horizontally sectioned in four animals. The 480-µm interval sections of the brain were stained with 0.2% cresyl violet solution  and subjected to immunohistochemistry to demonstrate the structure of the dura mater and the distribution of GAL and other substances within it.Eight male Wistar rats were used to determine the distribution of GAL-ir nerve fibers in the dura mater. They were anesthetized with isoflurane  until respiration was markedly suppressed and transvascularly perfused with 50 mL saline followed by 500 mL Zamboni fixativeTo analyze the expression of GAL and its relationship to CD11b and ED1 (markers for macrophages and dendritic cells), protein gene product 9.5 , and CGRP, single, double and triple immunofluorescence methods were used for the rat dura mater  for 60 min and avidin–biotin-complex prepared by VECTASTAIN Elite ABC Reagent (Vector) for 60 min. Then, sections were incubated with fluorescence-tyramide working solution for 10 min. After checking for GAL immunofluorescence, double and triple immunofluorescence were performed to demonstrate the relationship of GAL to CD11b, ED1, and CGRP. The GAL-stained sections were incubated with mouse anti-CD11B or anti-ED1 IgG and guinea pig anti-CGRP serum. The sections were further incubated with rhodamine red™-X or aminomethylcoumarin-conjugated donkey antiserum against guinea pig IgG or mouse IgG serum. For GAL and CD11b or PGP9.5, GAL-stained sections were incubated with rhodamine red™-X-conjugated donkey anti-rabbit IgG serum. After confirming no immunofluorescence by the secondary antiserum and rabbit anti-GAL serum, the sections were incubated with rabbit anti-CD11b and anti-PGP9.5 serum, followed by rhodamine red™-X-conjugated donkey anti-rabbit IgG serum. Then, 4′,6-diamidino-2-phenylindole  was applied to some immunofluorescence-stained sections as a nuclear counterstain.50. To check inappropriate reaction with the primary and secondary antibodies, TSA and double immunofluorescence methods were performed without either one or all of primary antibodies. The control sections showed only appropriate fluorescence. In addition, CD11b antibody was absorbed by the respective molecule (20 µg/ml). CD11b fluorescence cannot be detected in the absorption control.All procedures were conducted at room temperature. Sections were viewed with a conventional fluorescence microscope  or a confocal laser scanning microscope . The specificity of antibodies against GAL, CGRP, CD11b, ED1, and PGP9.5 has been described elsewhere53. The average of such immunoreactive density was recorded for each animal. Difference among several regions was examined by analysis of variance and Tukey’s test using the JMP® 16 statistical software .For the morphometric data, the brain was divided into three portions: the rostral 1/3, middle 1/3, and caudal 1/3 portions. Three to twelve sections were randomly selected from each of these portions in each of the four animals. The total length of the GAL-ir nerve fibers within the dura mater was measured in each of the various regions with a personal computer , a digital pencil , and a digital tracing tablet (Wakom) by freehand drawing . Data about the cerebral falx, other cerebral portions, and cerebellar dura mater were obtained from 1, 3, and 14 digital images (each image size = 660 µm × 820 µm), respectively, in each section. The proportion of their length to the area of each region was calculated for immunoreactive density7.To demonstrate the origin of GAL-ir nerve fibers in the dura mater, four male rats were used. Under deep anesthesia by i.p. injection with a mixture of 0.15 mg/kg medetomidine , 2.0 mg/kg midazolam , and 2.5 mg/kg butorphanol , the animal was put on the operative table, and the midline incision of the cranial skin was made at the back of its head. A small hole (diameter = 3 mm) was made on the lambda of the cranial bone with a dental round bar . After washing out small bone fragments with saline solution, the excess saline solution, blood, and exudate fluid were wiped out with a dry cotton. Then, a crystal of FG  with a diameter of 0.5 mm was placed on the middle portion of the tentorium cerebelli. The fluorescent tracer on the dura mater was sealed with α-cyanoacrylate . The hole on the cranial bone was covered with the skin, and the skin was subsequently stitched with a 4–0 silk suture 42. The TG and superior cervical ganglion were dissected and frozen-sectioned at 8 µm thickness. The sections of the TG were processed for co-expression of GAL and CGRP as described above. To determine the proportion of GAL- and CGRP-ir neurons among dura mater neurons, all FG-positive neurons were analyzed in every fifth section of the serial sections of the TG. For cell size analysis of GAL- and CGRP-ir neurons innervating the dura mater, the cross-sectional area of FG-positive cell bodies containing nuclear profiles was measured (Lumina Vision)54.After 3 days, the animals were re-anesthetized with the same mixture, and transvascularly perfused with Zamboni fixative


The polyacrylonitrile (PAN) nanofiber membrane was prepared by the electrospinning technique. The nitrile group on the PAN nanofiber surface was oxidized to carboxyl group by alkaline hydrolysis. The carboxylic group on the membrane surface was then converted to dye affinity membrane through reaction with ethylenediamine (EDA) and Cibacron Blue F3GA, sequentially. The adsorption characteristics of lysozyme onto the dye ligand affinity nanofiber membrane (namely P-EDA-Dye) were investigated under various conditions . Optimum experimental parameters were determined to be pH 7.5, a coupling concentration of EDA 40 μmol/mL, and an immobilization density of dye 267.19 mg/g membrane. To understand the mechanism of adsorption and possible rate controlling steps, a pseudo first-order, a pseudo second-order, and the Elovich models were first used to describe the experimental kinetic data. Equilibrium isotherms for the adsorption of lysozyme onto P-EDA-Dye nanofiber membrane were determined experimentally in this work. Our kinetic analysis on the adsorption of lysozyme onto P-EDA-Dye nanofiber membranes revealed that the pseudo second-order rate equation was favorable. The experimental data were satisfactorily fitted by the Langmuir isotherm model, and the thermodynamic parameters including the free energy change, enthalpy change, and entropy change of adsorption were also determined accordingly. Our results indicated that the free energy change had a negative value, suggesting that the adsorption process occurred spontaneously. Moreover, after five cycles of reuse, P-EDA-Dye nanofiber membranes still showed promising efficiency of lysozyme adsorption. Electrospun nanofiber membranes have been intensively researched in fields , such asDesirable features of nanofiber membranes intended for use in protein adsorption include low non-specific adsorption, high adsorption capacity, high adsorption rate, good biocompatibility, and high chemical/mechanical stability; these characteristics can be achieved by manipulation of chemical composition and synthesis operation that result in high porosity, large specific adsorption area, and good hydrophilicity of nanofiber membranes ,11,12,13Immobilization of dye ligand onto the PAN-based nanofiber membranes has been proven effective in selective adsorption of target proteins ,12,13. AChicken egg white (CEW) is composed of a variety of proteins including ovalbumin (~54%), ovotransferrin ~12–13%), ovomucoid (~12–13%), and lysozyme (~3.5%). CEW is considered as the main source of lysozyme, which is a commercially valuable biocatalyst  with sub–13%, ovoG°), enthalpy (ΔH°) and entropy (ΔS°)) [While the adsorption characteristics of lysozyme on the dyed nanofiber membranes have been investigated ,12,13, tMw = 120,000 g/mol, containing 93% acrylonitrile and 7% vinylacetate) was obtained from Fortune Industries Inc. . Polyethyleneterephthalate (PET) spunbond fabric  was supplied from Freudenberg Far Eastern Spunweb Co. . 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(N-Morpholino) ethane sulfonic acid hydrate (MES), N,N-dimethylacetamide (DMAc), and ethylenediamine dihydrochloride (EDA 2HCl) were obtained from Sigma-Aldrich . All other chemicals and solvents were used without further purification. Electrospinning device was purchased from Falco Tech Enterprise Co., Ltd. . CB reactive dye  was sourced from the First Chemical Manufacture Co., Ltd. .Polyacrylonitrile (PAN) yarn (2), which was pre-wrapped around the grounded steel drum. The PAN membrane was subjected to heat pressing process using a flat sandwich stainless steel plate at 373 K for 1 h. After the heat pressing process, the PAN-PET-PAN membrane became dense and entangled together.The electrospinning process of PAN nanofiber membrane, as shown in 2 (i.d. 2.5 cm), before being placed in 3 M NaOH at 353 K for 17 min. After alkaline treatment, the resultant carboxylated nanofibrous membrane (P-COOH) was continuously washed with distilled water to remove the residue NaOH. The membrane was then treated with 0.1 M HCl, followed by drying in an oven at 333 K before use.The PAN nanofiber membrane was first cut into 4.19 cm2O to remove the free EDA, and then dried in an oven at 333 K. The amount of EDA on the membrane was determined by measuring the initial and final EDA concentrations in the liquid phase.A piece of P-COOH nanofiber membrane was submerged into 5 mL of 0.1 M MES solution containing 200 μmol EDC and EDA 2HCl (pH 6.0). The mixture was shaken at 100 rpm and 298 K for 3 h. Finally, the aminated nanofiber membrane (P-EDA) was taken out, washed with H2) was placed in a flask containing 0.5–4.0 CB dye mg/mL dye and 20% NaCl in 5 mL 0.1 M Na2CO3 at pH 11. The reaction mixture was then heated at 333 K for 6 h in a sealed flask. The dyed P-EDA nanofiber membrane (P-EDA-Dye) were thoroughly washed with a series of solutions  until no dye molecule was detected in the washing solution. The dye concentration in solution was measured by spectrophotometer at 620 nm [A piece of P-EDA nanofiber membrane , sodium phosphate buffer , and glycine NaOH buffer . The initial concentration of lysozyme in each buffer solution was 0.8 mg/mL. The adsorption experiments were performed at 298 K and 100 rpm for 3 h. After this period had elapsed, the amount of lysozyme adsorbed on the membrane (mg lysozyme/g membrane) was determined by measuring the initial and equilibrium concentrations of lysozyme in the adsorption buffer. Different sets of batch adsorption studies were performed to investigate the effects of EDA concentration (20–200 μmol/mL), dye (0.1–4.0 mg/mL), sodium chloride concentration (0.1–1.0 M) during the adsorption process at 298 K. The effect of temperature on lysozyme adsorption was studied in 20 mM sodium phosphate buffer  with various lysozyme concentrations (0.1–1.0 mg/mL) at different temperatures (278–308 K). The lysozyme concentration in liquid phase was determined using the absorbance measured at 280 nm and the extinction coefficient of 2.65 (mL/mg·cm) . All thet, the samples of supernatants were collected from the flask for the determination of lysozyme concentration. The amount of adsorbed lysozyme on the membrane phase (mg lysozyme/g membrane) at time t was calculated according to the following equation:oC and tC (mg lysozyme/mL solution) are the initial lysozyme concentration before adsorption process and the lysozyme concentration at time t of adsorption process, respectively. tq (mg lysozyme/g membrane) is the concentration of lysozyme in the membrane solid phase at time t. v is the volume of aqueous phase (mL) and w is the weight of membrane (g). For the equilibrium studies in batch mode, a range of concentration of lysozyme (0.1–1.0 mg/mL) was made up in 5 mL of 20 mM sodium phosphate buffer, pH 7.5 in separate flasks. The flasks were placed in a shaker at different temperatures (278–308 K) at 100 rpm for 3 h. The amount of adsorbed lysozyme on the membrane phase at equilibrium was calculated by Equation (1).In the kinetic adsorption experiment, various parameters were investigated in batch mode, including adsorption pH (4–11), immobilized dye concentration (0.5–4.0 mg/mL), EDA concentration used in nanofiber membrane fabrication (100–1000 μmol/5 mL), and temperature of adsorption experiments (278–308 K). In brief, a piece of P-EDA-Dye nanofiber membrane (approximately 30 mg) was added to a flask containing 5 mL of lysozyme solution. The flasks were sealed and placed in a shaker at 100 rpm for 3 h. At the designated time A simple adsorption kinetic model, the pseudo first-order kinetic model, can be described by Equation (2) ,24:(2)dqIntegration of Equation (2) results in Equation (3):eq−qt)ln. In this adsorption kinetic model, the transport of protein to the interface of membrane is assumed to be the rate-limiting step.The linear plots of The pseudo second-order kinetic model can be described by Equation (3) ,24:(4)dqIntegration of Equation (4) yields Equation (5):k2) and equilibrium binding capacity (eqq) can be obtained from the intercept and slope of the plot of (tt/q) vs. t, respectively. When using this adsorption kinetic model, the surface reaction is assumed to be the rate-limiting step.The overall rate constant  :(6)dqtdtα is the initial sorption rate (mg/g·min) and the parameter β is related to the extent of surface coverage and activation energy for chemisorption (g/mg).Elovich equation was derived via an integration of the rate equation using the same boundary conditions as the pseudo first- and second-order equation.ik is the intra-particle diffusion rate constant, mg/g·min0.5. ik values under different conditions were determined from the slopes of straight-line portions of the respective plots.An intra-particle diffusion-based model, which can be described by Equation (8), was used to investigate the mechanism of the adsorption of lysozyme on the P-EDA-Dye nanofiber membrane .(8)qt=kIn general, the adsorption mechanism could involve several steps in which one or any of their combination would control the rate of adsorption. There are three steps involved: (1) the mass transfer across the external boundary layer film of liquid surrounding the outside of the particle, (2) the adsorption at a site on the internal/external surface, which is often assumed to be very rapid, (3) the diffusion of target molecules to an adsorption site by diffusion either through the liquid-filled pores or the solid surface.C (mg lysozyme/mL solution) is the equilibrium concentration of lysozyme in the aqueous phase. q (mg lysozyme/g-membrane) is the equilibrium lysozyme concentration in the membrane solid phase. maxq is the maximum binding capacity of membrane for lysozyme (mg lysozyme/g) and dK is the dissociation constant of lysozyme-membrane complex (mg lysozyme/mL solution). The Langmuir constants, maxq and dK, were evaluated from the corresponding semi-reciprocal plot (C/q vs. C) of the linear Equation (10).Adsorption isotherms studies have been conducted to characterize the adsorption of lysozyme by the membrane . The adsdK (mg/mL) denotes the binding strength between lysozyme and membrane matrix and is considered an inverse measure of the equilibrium association constant  for the binding between the membrane and lysozyme.The value of FK and n can be related to binding capacity and adsorption intensity, respectively, and are determined from the straight-line plots of ln(q) vs. ln(C) for the adsorption of lysozyme as described in Equation (12).The Freundlich model assumes that the biomolecule adsorption takes place on a heterogeneous surface through a monolayer adsorption ,23. Thisb is related to the heat of adsorption (J/mol), R is gas constant (8.314 J/mol/K), T is the temperature (K), and TK is equilibrium binding constant (mL/g). These constants were determined from, a plot of q versus ln(C).The Temkin isotherm assumes that the heat of adsorption decreases linearly rather than logarithmically due to the adsorbate/adsorbent interaction, as implied in the Freundlich equation. The linear form of Temkin isotherm is given in the following equation ,23:(13)q−1 in P-COOH, P-EDA, P-EDA-Dye nanofiber membranes are attributed to the -OH stretch from the alcohols and phenols groups [−1  are vibrational signal generated from cyanate group (-CN) [−1 (-OH stretch), 1640–1560 cm−1 (-N=N-), and 820 cm−1 (-COO) are the manifestations from the absorption of -NH2 groups available on the material [2) groups on the P-EDA nanofiber membrane were ~392 μmol/g, based on the reaction of the negatively charged AO7 dye with the positive surface functional group (-NH3+). Similarly, the positively charged TBO dye reacted with the negative surface ions (-COO−) whereby the number of carboxyl groups on the P-COOH nanofiber membrane was ~423 μmol/g.FTIR spectra of PAN, P-COOH, P-EDA, and P-EDA-Dye nanofiber membranes show numerous peaks which indicate their chemical structures . The bros groups ,13. The up (-CN) . Furthermaterial . The amiThe surface morphological attributes of PAN, P-COOH, P-EDA, and P-EDA-Dye nanofiber membranes are shown in The binding capacity of the P-EDA-Dye nanofiber membrane for lysozyme under various operating parameters  was investigated. The results are shown in I = 11). Here, the positively charged groups may be present in specific region of the lysozyme molecule, although there exists more positively charged amino groups under a lower pH condition [Studies of the adsorption of lysozyme by P-EDA-Dye nanofiber membrane were carried out in the adsorption pH ranging from 4 to 10. As observed from ondition . Similarondition ,13. AlthI value of lysozyme), the binding capacity of P-EDA-Dye nanofiber membrane decreased to ~200.81 mg/g, which may be due to less positively charged groups on the lysozyme. This contributes to a less pronounced electrostatic effect on the positively charged lysozyme and the negatively charged P-EDA-Dye nanofiber membrane (3 units of SO3Na). Our results suggested that the optimal pH for lysozyme adsorption on P-EDA-Dye nanofiber membrane is pH 7.5.As the adsorption pH was increased to 10 (close to the p2) and/or weak anion (-NH3+) exchanger. The P-EDA nanofiber membrane may either repel or attract protein molecules depending on their pI values and the degree of positive charge on their surface. In addition, the adsorption process of lysozyme with polyelectrolytic characteristics may be controlled by the molecular size and flexibility of lysozyme, diffusion behavior of lysozyme in both bulk solution and membrane pores, and strength of hydrophilic and/or hydrophobic forces existing between the lysozyme and the membrane [The EDA-modified nanofiber membrane contains a high degree of amine groups serving as a weak cation , the binding capacity of P-EDA-Dye nanofiber membrane for lysozyme ranged in between 136.37 mg/g and 258.05 mg/g, as shown in Since large amounts of salts are generally utilized in the elution process of the adsorbed lysozyme from the membrane ,13, the eq,expq increased from 235.96 to 373.73 mg lysozyme/g when the temperature raised from 278 K to 308 K. Since the adsorption rate of hydrophobic or specific affinity interaction would increase as the temperature increases [The rate of adsorption of lysozyme onto the P-EDA-Dye nanofiber membrane was investigated at varying temperatures from 278 K to 308 K, and the results are shown in ncreases , the mecncreases .3− groups), the mechanism of adsorption of lysozyme may be an ion exchange process. The dyed nanofiber membrane, +(P-EDA-Dye)−Na, has the single +Na ion balancing the charge of one dye immobilized composite membrane in the membrane framework, lysozyme molecule (namely 3+P-NH) is the positively charged group at pH adsorption pH 7.5, and −-(NH3+-P)(P-EDA-Dye) represents charge–charge interactions between the immobilized lysozyme and dyed membrane. Comparison of the experimental adsorption capacities of the P-EDA-Dye nanofiber membrane for lysozyme and their theoretical estimates are presented in 2 > 0.98) were obtained when using the pseudo second-order kinetic model  were found to be close to the experimental eq,expq values .To investigate the governing mechanism of the adsorption process, pseudo first-order and pseudo second-order kinetic models were used to analyze the dynamic experimental data and to determine the adsorption rate constants. Due to the cationic characteristics of P-EDA-Dye nanofiber membrane  was reported to follow the Arrhenius equation (Equation (14)) [The temperature dependence of overall rate constant (on (14)) .(14)k2=aE) for lysozyme adsorption onto the P-EDA-Dye nanofiber membrane was ~2389.37 cal/mol. The overall rate constant, k2, was found to increase from 2.67 × 10−4 to 3.71 × 10−4 mg lysozyme/min g when the temperature increases from 278 K to 308 K in the adsorption of lysozyme.The results were illustrated as an Arrhenius plot in R2) are relatively high (approximately 0.97), and the results are shown in tq versus t0.5 at different temperatures presented multi-linearity relation, indicating more than one process affecting the lysozyme adsorption. The ik values under different operating conditions were calculated from the slopes of the straight-line portions of the respective plots. The plot of tq versus t0.5 presents the multi-linearity, indicating that two or more pore diffusion steps occur in the adsorption process [As shown in  process ,23. Moreik and I (intercept) obtained from each stage linear regression analysis are listed in i,k1) > the second linear portion . The changes in ik values could be attributed to the adsorption stages of the external diffusion and internal channel or micro-pore diffusion, respectively. From the pore size distribution, P-EDA-Dye nanofiber membrane has an average pore size of 0.45 µm and the pores are randomly distributed over the entire membrane surface. Hence, the multistage adsorption processes would be observed in these cases. The second portion of the plot seemed to refer to the channel diffusion into micro-pores as the rate-limiting step in the adsorption process on the membrane.As can be seen from R2 > 0.99) was obtained using the Langmuir isotherm, suggesting that the Langmuir isotherm was more adequate in fitting the experiment equilibrium data, as compared to the Freundlich model (R2 = 0.8541–0.9836) and Temkin model (R2 = 0.8918–0.9878). As shown in dK) of P-EDA-Dye nanofiber membrane decreased from 0.0506 mg lysozyme/mL solution to 0.0845 mg lysozyme/mL solution. As the temperature rose from 278 to 308 K, the maximum binding capacity  of P-EDA-Dye nanofiber membrane increased from 262.19 to 407.66 mg/g. The observed elevation in binding capacity may be attributed to the chemical interaction between lysozyme and membrane, or the production of some new binding sites at higher temperatures. In comparison to P-CS-Dye membranes  [max,expq of P-CS-Dye nanofiber membrane was 245.58 mg lysozyme/g at 298 K. The dK value decreased with an increase in temperature, indicating that the adsorption of lysozyme onto nanofiber membranes was favorable at a higher temperature.To explore the equilibrium behavior of lysozyme adsorption onto the nanofiber membranes, three isotherm models including Langmuir, Freundlich, and Temkin isotherms were used to fit against the equilibrium adsorption experiment data ,23. Figuhitosan) , the P-EG°, ΔH°, and ΔS°), lysozyme was adsorbed onto the P-EDA-Dye nanofiber membrane at different temperatures ranging from 278 K to 308 K. The magnitudes of thermodynamic parameters  were determined using the equations below [To calculate the thermodynamic parameters ΔadsK on 1/T for the adsorption of lysozyme onto P-EDA-Dye nanofiber membrane, along with the corresponding Van ’t Hoff plot, are shown in adsln K vs. 1/T multiplied by −R  equals to The dependence of the equilibrium association constant membrane . The estThe possibility of reusing P-EDA-Dye nanofiber membrane for multiple cycles of lysozyme adsorption was explored in this study. The adsorption–desorption operations in batch mode were repeated for five cycles using the same P-EDA-Dye nanofiber membrane as shown in eqq values are difficult to be determined experimentally. From our experimental data, the activation energy for lysozyme adsorption onto the P-EDA-Dye nanofiber membrane was determined to be ~2389.37 cal/mol at the temperature range of 278–308 K. The isotherm curves correlated well with the Langmuir model. The binding capacity decreased with increasing buffer ionic strength. This may be due to the presence of electrostatic interaction between dyed membrane and lysozyme. The interaction may play an important role in the adsorption process of lysozyme. In addition, our results showed that the values of ΔG° were negative, indicating the adsorption process to be a spontaneous reaction. The negative value of ΔH° showed that the adsorption was an exothermic process and the positive values of ΔS° indicated an increase randomness at the solid–liquid interface during lysozyme adsorption. Further research will be needed to demonstrate that CB dyes immobilized on the nanofiber membrane may serve as an alternative technique for the purification of lysozyme by membrane adsorption chromatography.The kinetics of adsorption of lysozyme onto the P-EDA-Dye nanofiber membrane was studied experimentally and theoretically under different operating conditions. The values of kinetic parameters associated with the adsorption mechanism of lysozyme were determined in a batch stirred tank system. The results showed that the pseudo second-order kinetic model gave the best correlation of the experimental data for all operating conditions used in this study. The surface reaction was proposed to be the rate-limiting step for the adsorption of lysozyme onto the membrane. Moreover, the pseudo second-order kinetic model could estimate the maximum binding capacity for lysozyme, since the


Staphylococcus aureus (MRSA) remains one of the leading causes of both nosocomial and community infections worldwide. In the Philippines, MRSA rates have remained above 50% since 2010, but resistance to other antibiotics, including vancomycin, is low. The MRSA burden can be partially attributed to pathogen-specific characteristics of the circulating clones, but little was known about the S. aureus clones circulating in the Philippines.Methicillin-resistant S. aureus isolates collected in 2013–2014 within the Antimicrobial Resistance Surveillance Program. The multilocus sequence type, spa type, SCCmec type, presence of antimicrobial resistance (AMR) determinants and virulence genes and relatedness between the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined.We sequenced the whole genomes of 116 spa-t019-SCCmec-IV-PVL+, CC5-SCCmec-typeIV and ST239-spa-t030-SCCmec-typeIII. The CC30 genomes were related to the South-West Pacific clone but formed a distinct, diverse lineage, with evidence of global dissemination. We showed independent acquisition of resistance to sulfamethoxazole/trimethoprim in various locations and genetic clones but mostly in paediatric patients with invasive infections. The concordance between phenotypic and genotypic resistance was 99.68% overall for eight antibiotics in seven classes.The MRSA population in the Philippines comprised a limited number of genetic clones, including several international epidemic clones, such as CC30-We have made the first comprehensive genomic survey of S. aureus in the Philippines, which bridges the gap in genomic data from the Western Pacific Region and will constitute the genetic background for contextualizing prospective surveillance. Staphylococcus aureus (MRSA) remains one of the leading causes of both nosocomial and community infections worldwide. .Methicillin-resistant mec (SCCmec) that carries the mecA or mecC gene, which confers resistance to methicillin and most β-lactam antibiotics. Importantly, some MRSA clones have also acquired resistance to vancomycin, the first-line antibiotic treatment for severe MRSA infections in hospitals,  being ST30 , ST239 , ST5 , ST59  and ST72 (Republic of Korea).  of the Philippines Department of Health during the period January 2013 to December 2014. Isolates found to be resistant to oxacillin (i.e. MRSA) were subsequently referred to the ARSP reference laboratory for confirmation. Of the 412 and 384 isolates referred in 2013 and 2014, respectively, a total of 118 MRSA isolates from 17 sentinel sites were selected for whole-genome sequencing (WGS) on the basis of their resistance profile . The zone of inhibition and the minimum inhibitory concentration of antibiotics were interpreted according to the 26th edition of the Clinical and Laboratory Standard Institute guidelines. . DNA extracts were multiplexed and sequenced on the Illumina HiSeq platform  with 100-bp paired-end reads. Raw sequence data were deposited in the European Nucleotide Archive under study accession No. PRJEB17615. Run accessions are provided on the Microreact projects.S. aureus subsp. aureus strain TW20 (accession FN433596), as previously described.  were determined from assemblies with Pathogenwatch (https://pathogen.watch/) or from sequence reads with ARIBA  or ILRI_Eymole1/1 (NZ_LN626917) with the Burrows Wheeler aligner (BWA) v0.7.12, as described in detail previously.  30 phylogeny, recombination regions detected with Gubbins (S. aureus genomes available at the European Nucleotide Archive with geolocation and isolation date was inferred by an approximately maximum likelihood phylogenetic method with FastTree. (Evolutionary relations among isolates were inferred from single nucleotide polymorphisms (SNPs) by mapping the paired-end reads to the reference genomes of lukF-PV and lukS-PV genes were identified from raw sequence reads with ARIBA  http://microreact.org).All project data, including inferred phylogeny, AMR predictions and metadata, were made available through the web application Microreact  of the isolates were from patients aged < 1 year (n = 66) and 44% from females (n = 50). As invasive isolates were prioritized, the most common specimen source was blood , followed by wounds . The majority of the infections  were classified as community-associated MRSA.Of the 118 MRSA isolates submitted for WGS, 116 were confirmed as blaZ and mecA genes , oxacillin (mecA), cefoxitin (mecA), gentamicin (aacA-aphD), erythromycin , clindamycin (ermC), tetracycline , ciprofloxacin and levofloxacin , chloramphenicol (catA1) and rifampicin (rpoB_H481N). The IleS gene that confers resistance to mupirocin and the sdrM gene conferring resistance to norfloxacin were identified in three and 23 isolates, respectively; however, mupirocin was not tested in the laboratory, and norfloxacin was tested only against isolates from urine specimens. Hence, these two antibiotics were not included in the concordance analysis.Isolates were tested for susceptibility to 14 antibiotics in eight classes. All the isolates were susceptible to vancomycin and linezolid and resistant to penicillin, oxacillin and cefoxitin, consistent with the presence of the mecA resistance gene. Conversely, one isolate was falsely predicted to be resistant to sulfamethoxazole/trimethoprim on the basis of the presence of the dfrG gene.Comparisons between phenotypic and genotypic data are presented for eight key antibiotics in seven classes  of the isolates belonged to clonal complex (CC) 30, distributed between ST30 (n = 81), its single-locus variant ST1456 (n = 2) and three ST30 genomes showing novel aroE (n = 2) and yqiL (n = 1) alleles. CC5 was represented by nine genomes and ST834 by six. The most prevalent of the 29 different spa types identified was t019 (62%), which coincided with genomes assigned to CC30. The spa types identified for the CC5 genomes were t002 (n = 5), t105 (n = 3) and t067 (n = 1). Most of the SCCmec cassettes identified in the genomes belonged to type IV , followed by type III . The SCCmec type could not be determined for four genomes. The numbers and most common ST and spa types found in each of the sentinel sites are shown in spa-t019-SCCmec-IV was the most prevalent MRSA clone in this retrospective collection .MLST, Fig. 2A), in agreement with the clonal population previously described for S. aureus. , none of which displayed a strong phylogeographical signal. Both genes lukS-PV and lukF-PV that encode the PVL were found in 75 of the 86 CC30 genomes , indicating that the majority (87.2%) are PVL-positive .The phylogenetic tree shows that the population was composed of discrete clades that matched the ST distribution and were separated by long branches (mec-typeIV (71%) were from paediatric patients (compared with 20% of the entire data set) and were generally clustered according to their spa type; however, they displayed no clear phylogeographical distribution. Two isolates from different, distant locations carried both lukS-PV and lukF-PV genes (PVL-positive). The two ST239 isolates were from the same patient, spa type t030, SCCmec-typeIII, PVL-negative and multidrug-resistant.Two additional epidemic clones were identified, CC5 and ST239 (CC8). Six of the nine CC5-SCCn = 8) and an abscess (n = 1) and, interestingly, mainly from paediatric patients .The nine isolates with resistance to sulfamethoxazole/trimethoprim were from four locations  and belonged to four different clones , which suggests that resistance to this antibiotic has emerged independently  and the United States of America (n = 3241) and the epidemic clones ST8 (n = 2343), ST22 (n = 1526) and ST5 (n = 720) prevalent in those regions . Health-care-associated EMRSA-15 (ST22) was notably absent from our collection, as was livestock-associated CC398 . The Philippine ST5 genomes did not form a monophyletic group within the CC5 clade, suggesting more than one origin. CC30-spa-t019-SCCmec-IV-PVL+ MRSA genomes from the Philippines formed a discrete cluster within ST30 with small numbers of genomes from the United States of America (n = 5), the United Kingdom of Great Britain and Northern Ireland (n = 3) and Germany .We placed the genomes from our retrospective collection in the global context of 7821 contemporary Fig. 3B). In addition, the genomes from the Philippines clustered with genomes from Argentina, Germany, the United Kingdom and the USA , indicating that the epidemic diversification from the South-West Pacific clone was accompanied by global dissemination.Several successful pandemic clones have emerged within CC30, such as the methicillin-sensitive phage type 80/81, (S. aureus collections in Europe and the United Kingdom, (In this study, we combined WGS and laboratory-based surveillance to characterize MRSA circulating in the Philippines in 2013 and 2014. High levels of concordance between phenotypic and genotypic resistance were observed for all the antibiotics tested. This was previously reported for spa-t019-SCCmec-IV-PVL+. Community-acquired CC30-spa-t019-PVL+ MRSA was previously reported in one hospital in the Philippines between 2004 and 2006 and from other countries in South-East Asia, with potential clonal expansion. (Few studies of the molecular epidemiology of MRSA in the Philippines have been published. We found that the MRSA population in 2013 and 2014 comprised a limited number of genetic lineages, dominated by CC30-S. aureus in the Philippines, bridging the gap in genomic data from the Western Pacific Region, and provides the genetic background for contextualizing prospective surveillance for infection control.Genotypic characterization of circulating MRSA strains, with phenotypic and epidemiological data, led to the identification of several global epidemic clones and revealed the lack of a strong phylogeographic structure in the population from patients admitted to health-care facilities in a country with a high burden of MRSA. This supports interventions to reduce the burden of disease in the general population. (